γ-2 and GSG1L bind with comparable affinities to the tetrameric GluA1 core

Background The AMPA-type ionotropic glutamate receptor mediates fast excitatory neurotransmission in the brain. A variety of auxiliary subunits regulate its gating properties, assembly, and trafficking, but it is unknown if the binding of these auxiliary subunits to the receptor core is dynamically regulated. Here we investigate the interplay of the two auxiliary subunits γ-2 and GSG1L when binding to the AMPA receptor composed of four GluA1 subunits. Methods We use a three-color single-molecule imaging approach in living cells, which allows the direct observation of the receptors and both auxiliary subunits. Colocalization of different colors can be interpreted as interaction of the respective receptor subunits. Results Depending on the relative expression levels of γ-2 and GSG1L, the occupancy of binding sites shifts from one auxiliary subunit to the other, supporting the idea that they compete for binding to the receptor. Based on a model where each of the four binding sites at the receptor core can be either occupied by γ-2 or GSG1L, our experiments yield apparent dissociation constants for γ-2 and GSG1L in the range of 2.0–2.5/µm2. Conclusions The result that both binding affinities are in the same range is a prerequisite for dynamic changes of receptor composition under native conditions. Supplementary Information The online version contains supplementary material available at 10.1186/s11658-023-00470-9.


Note 1: Rationale for choosing intracellularly injected nanobodies for labeling
For three-color single-molecule imaging, we needed three spectrally separated tags. Since GFP and mCherry cover the green and orange-red emission range, an additional color can be placed either in the blue or in the far-red range. Because the Olympus 100x NA 1.70 objective, which we use due to its high signal-to-noise ratio for single-molecule imaging, requires an immersion liquid that does not transmit wavelengths below 480 nm well, we could not use blue or cyan fluorescent proteins. There are no sufficiently bright fluorescent proteins in the far-red range that can be spectrally separated from mCherry, and therefore we resorted to using an organic dye.
One possibility would be the use of a tagging system like the SNAP-tag where an extracellular tag at the target protein can be labeled by a fluorescent substrate that is supplied through the medium. However, the Xenopus oocytes are covered by the vitelline membrane, a gel-like layer where the externally applied substrate gets entangled. Although the vitelline membrane is being removed immediately before placing the cell on the coverslip for imaging, it is impossible to effectively wash the cell because without the vitelline membrane, it is delicate and ruptures easily. On the other hand, for the planned experiments it is also not possible to resort to a different cellular expression system where extracellular labeling works well (e.g. mammalian cell lines). The reason is that in Xenopus oocytes, ionotropic glutamate receptors and some other membrane proteins remain immobile in the membrane (for unknown reasons), and therefore facilitate counting photobleaching steps of attached fluorescent proteins. In mammalian cells, these proteins are mobile, which makes it impossible to reliably count photobleaching steps. In addition, Xenopus oocytes allow the control of expression levels by adjusting the amount of injected RNA.
We therefore came up with the idea to use an epitope tagging system intracellularly and inject the substrate into the cell. As it is impossible to wash out the substrate after labeling, once it is injected into the oocyte, we searched for a tagging system that requires only a low concentration of substrate to minimize the background, and chose the anti-GFP nanobody, which binds to GFP with an affinity below 1 nM and has previously been used for single-molecule imaging of membrane proteins in mammalian cells [13−15].
A647-Nb contained a total of 4 solvent-exposed lysines that could potentially have been labeled with dye NHS esters, and had an average of 1.0−1.5 A647 dye labels attached; assuming all solventexposed lysines have an equal chance of getting labeled, the number of labels per A647-Nb molecule should be binomially distributed. Accordingly, determining the subunit number of the target protein by counting the photobleaching steps from the dye is difficult.

Note 2: Model for binding of -2 and GSG1L to GluA1
To model the binding of -2 and GSG1L to GluA1, we assume that the densities are in an equilibrium for all assemblies. From Fig. 5A  Because not all fluorescent protein molecules are functional, the bleaching steps counts do not equal the actual numbers of auxiliary subunits. We defined the probability of mNeonGreen to be functional as and of mCherry as . Since the receptors contained four GluA1-GFP(Y66L) and were usually labeled with multiple A647-Nb, we approximated that all receptor cores were visible (see Fig. 3C). Therefore, we could write the concentration of counted spots as:  With the above equations and the counted number of protein complexes, we could fit the and by least-square fitting. In practice, was set to 0.77, which was fitted from GluA1-mNeonGreen experiments, and was set to 0.65, which was our estimate from previous results and was also determined independently [17].